The protein serine/threonine phosphatase calcineurin (PP2B) is a signal integrator that converts the calcium signal into gene expression, affecting both vertebrate development and immune function, often through the NFAT family of transcription factors. Calcineurin is the well known target of the clinically important immunosuppressant cyclosporin A and FK506 which have been used to document the roles of calcineurin in many important biological processes. However, calcineurin's immediate substrates remain unidentified in many cases. Over the past several years, by taking structural and biochemical approaches, I have shown that calcineurin communicates with its substrates by recognizing a conserved sequence motif termed the PxIxIT motif. The PxIxIT motif is critical in maintaining the optimal calcineurin-binding affinity for a balanced signal output. A peculiar quality of the PxIxIT motif is that each residue contributes independently to binding regardless of the sequence context, and the total affinity of binding can be estimated by a summation of the individual contributions. Here, I propose to take advantage of the unique property of the PxIxIT motif to efficiently scan the entire human proteome for all PxIxIT motif-containing calcineurin-binding proteins. In Aim 1, I will screen several panels of about 100 peptides each that are variants of a PxIxIT sequence of known affinity (the reference peptide). As explained in detail in the proposal, these data will allow me to predict the affinity of any given peptide for the PxIxIT-binding site of calcineurin;in Aim 2, a collection of the PxIxIT sequences predicted to bind calcineurin with physiological relevant affinities will be used in an extensive human proteome database search. Candidate proteins that pass a set of further criteria will be studied in Aim 3;in Aim 3, I will conduct a series of experiments to confirm if these candidate proteins are indeed calcineurin substrates. I expect that it will be possible to identify most of the hidden targets of calcineurin in the human genome using this novel approach. My long-term goal is to define these uncharacterized calcium/calcineurin signaling pathways and reveal any link they may have to human diseases. PUBLIC HEALTH RELEVANCE: Signaling through calcium controls diverse cellular functions, and calcium dysregulation can lead to severe physiological outcomes. As an essential protein serine/threonine phosphatase, calcineurin is a key component in the calcium signaling network and plays critical roles in both vertebrate development and immune function. Aberrant activity of calcineurin is implicated in a myriad of human diseases, including hypertrophic heart disease, muscular dystrophies, schizophrenia, Down's syndrome, Alzheimer disease, osteoporosis, and autoimmune diseases such as rheumatoid arthritis and cancer. Calcineurin recognizes its substrates through direct "docking" at a conserved sequence motif termed "PxIxIT" motif. As of today, only about a dozen of PxIxIT motif-containing substrates of calcineurin are known to us. Given the important roles calcineurin plays and a wide range of pathological conditions calcineurin is involved in, it is imperative to uncover those hidden calcineurin targets and signaling pathways. Here, I propose a straightforward and feasible way to achieve this goal. A complete understanding of the biology of calcineurin signaling will offer us a wealth of opportunities and better strategies for more effective pharmacological intervention against human diseases.